No abstract

Microbial rhodopsins are membrane proteins found widely in archaea, eubacteria and eukaryotes (fungal and algal species). They have various functions, such as light-driven ion pumps, light-gated ion channels, light sensors and light-activated enzymes. A light-driven proton pump bacteriorhodopsin (BR) contains a DTD motif at positions 85, 89, and 96, which is unique to archaeal proton pumps. Recently, channelrhodopsins (ChRs) containing the DTD motif, whose sequential identity is ~20% similar to BR and to cation ChRs in Chlamydomonas reinhardtii (CrCCRs), were found. While extensive studies on ChRs have been performed with CrCCR2, the molecular properties of DTD ChRs remain an intrigue. In this paper, we studied a DTD rhodopsin from G. theta (GtCCR4) using electrophysiological measurements, flash photolysis, and low-temperature difference FTIR spectroscopy. Electrophysiological measurements clearly showed that GtCCR4 functions as a light-gated cation channel, similar to other G. theta DTD ChRs (GtCCR1-3). Light-driven proton pump activity was also suggested for GtCCR4. Both electrophysiological and flash photolysis experiments showed that channel closing occurs upon reprotonation of the Schiff base, suggesting that the dynamics of retinal and channels are tightly coupled in GtCCR4. From Fourier transform infrared (FTIR) spectroscopy at 77 K, we found that the primary reaction is an all-trans to a 13-cis photoisomerization, like other microbial rhodopsins, although perturbations in the secondary structure were much smaller in GtCCR4 than in CrCCR2.

Definition of rhodopsin is the retinal‐binding membrane protein with the Schiff base linkage at a lysine on the 7th transmembrane helix. However, ~ 600 microbial rhodopsins lack retinal‐binding lysine at the corresponding position (Rh‐noK) among ~ 5500 known microbial rhodopsins, suggesting that Rh‐noK has each functional role without chromophore. Here, we report successful functional recovery of Rh‐noK. Two Rh‐noKs from bacteria were heterologously expressed in Escherichia coli, which exhibited no color. When retinal‐binding lysine was introduced, one of them gained visible color. Additional mutation of the Schiff base counterion further gained proton‐pumping activity. Successful engineered functional recovery such as visible color and proton‐pump activity suggests that the Rh‐noK protein forms a characteristic structure of microbial rhodopsins.

No abstract

The Cryptomonad Guillardia theta has 42 genes encoding microbial rhodopsin-like proteins in their genomes. Light-driven ion-pump activity has been reported for some rhodopsins based on heterologous E. coli or mammalian cell expression systems. However, neither their physiological roles nor the expression of those genes in native cells are known. To reveal their physiological roles, we investigated the expression patterns of these genes under various growth conditions. Nitrogen (N) deficiency induced color change in exponentially growing G. theta cells from brown to green. The 29 rhodopsin-like genes were expressed in native cells. We found that the expression of 6 genes was induced under N depletion, while that of another 6 genes was reduced under N depletion.